Phytochrome is a photoreceptor that control diverse aspects of growth and development in higher plants. Upon irradiation, the photoreceptor undergoes reversible interconversion between biologically inactive, red-absorbing phytochrome (Pr) and biologically active, far-red light absorbing phytochrome (Pfr) that enables it act as a molecular light switch. Photoconversion into Pfr form by red light treatment triggers its nuclear translocation from cytosol, initiating signaling that alters gene expression and thereby growth and development of plants. There are two photoisomers, red light (λmax=660 nm) absorbing form (designated to Pr) and far-red light (λmax=730 nm) absorbing form (designated to Pfr). Particularly, the absorption spectra of phytochrome are near the spectrum of canopy (FIG. 1, Neff et al, 2000). This spectral property shows it is directly related to shade avoidance. The initiation of shade avoidance depends on low R (red): FR (far red light) ratio. Low R: FR ratio accelerates not only the shade avoidance reaction that involves hypocotyls elongation, but also early flowering that causes immature fruit developments (Smith & Whitelam, 1997).
The photoreceptor consists of a 116-127 kDa apoprotein and a covalently attached linear tetrapyrrole chromophore. In plants, the apoproteins are encodes by a small gene family, e.g., five members PHYA-E in Arabidopsis. Molecular genetic analysis revealed that individual members of phytochromes play overlapping but distinct physiological roles. PHYA, a type 1 photo-labile phytochrome, controls very low fluence response and FR-high irradiance response, while type 2 phytochrome, encoded by PHYB-E, abundant in light-grown tissues, regulates low fluence responses (Quail et al., 1995; Neff et al., 2000).
Previously, oat PhyA was shown to undergo post-translational modification after red-light treatment, including phosphorylation at serine 598th residue (Lapko et al., 1999). The Pfr-specific phosphorylation at serine 598th residue suggested a regulatory role of this residue on photo-sensory signalling. To test the possibility, in the present invention, we performed site-directed mutagenesis with oat PHYA, substituting serine 598th to alanine (designated S598A PHYA in the invention). The biological activity of mutated PHYA was compared with wild type PHYA by overexpression into phyA-null mutant of Arabidopsis. Under FR light condition, both wild type PHYA and S598A PHYA could complement phyA-deficient mutant, showing FR-high irradiance response. However, at adult stage, transgenic Arabidopsis plants overexpressing S598A PHYA exhibited shortened internode in adult plants and shortened petiole, whereas transgenic plants overexpressing wild type PHYA did not show any noticeable defect in adult morphology. Overexpression of PP2A gene resulted in a suppressed internode phenotype similar to that of S598A mutant phytochrome. Thus, we include in the invention the overexpression of PP2A gene as being equivalent to bona fide hyperactive phytochrome by keeping it dephosphorylated in vivo. These results indicate that S598A PHYA is more biologically active than wild type PHYA at least in the regulation of internode elongation.
Serine-to-alanine substitutions at the N-terminal serine/threonine cluster in phytochromes result in hyperactive phytochromes in Arabidopsis thaliana (Stockhaus et al., 1992). Among the N-terminal serine residues, serine-7 is the only residue in the cluster that is specifically autophosphorylated or phosphorylated by a phytochrome kinase in vivo (Lapko et al., 1997). Thus, S7A mutant phytochrome is a hyperactive phytochrome.
It has been possible to locate the active site of the autophosphorylating phytochrome A (acting as a “phytochrome kinase”). The PAS-related domain in the C-terminal half of the protein contains active site residues. Mutation or deletion of these residues is expected to result in hyperactivity of phytochrome A in vivo, since such mutants cannot autophosphorylate the protein.
By using the method of site-directed mutagenesis (Bhoo et al., 1997) and DNA shuffling, we have also generated phytochrome A mutants that absorb far-red shade light more effectively than wild type. This was achieved by substituting critical amino acid residues (for example, isoleucine-80) within the chromophore binding crevice of phytochrome A. FIG. 1 illustrates how a few nanometer red shift of the Pr-absorption band, so that it can absorb canopy and shade lights several orders of magnitude more effectively in the far-red wavelength than with the overexpression of wild type phytochrome. We propose that the far-red spectral action spectrum for the induction of seed germination (Shinomura et al., 1996) is consistent with the Pr-absorption spectrum of “hot band” or “twisted” chromophore conformation origin, the bathochromic mutant phytochromes are hyperactive in the responses of higher plants to far-red light.
This invention can be practically applied to control growth and development in general and internode elongation and leaf greening of higher plants in particular (Smith and Whitelam, 1997). The higher plants referred to here are those economically important in agriculture and horticulture. As used herein, the term “economically important higher plants” refers to higher plants that are capable of photosynthesis and widely cultivated for commercial purpose. The term “plant cell” includes any cells derived from a higher plant, including differentiated as well as undifferentiated tissues, such as callus and plant seeds.